Biological samples of cells and/or tissues are obtained for purposes of analyzing the biological constituents that comprise the sample. The analysis may be a molecular analysis to determine if a particular analyte, such as an enzyme, protein, or nucleic acid, is present within the sample. The presence or absence of an analyte is frequently used to determine a disease state, such as cancer.
Prior to the analysis the biological sample must be prepared by appropriate methods, such as fixation, embedding, and sectioning. For example, a tissue biopsy may be removed from a patient and processed to adhere the tissue section or biological sample to a microscope slide. The biological sample is then stained in a manner to produce a colored stain on the tissue that corresponds to the analyte under evaluation. It is typical that the analyte will be located within various cells or structures distributed throughout the tissue. However the analyte density may vary greatly by location. In some instances it is desirable to know the analyte density level at the various locations.
Two common staining methods are in situ hybridization (ISH) for evaluation of nucleic acid analytes and immunohistochemistry (IHC) for evaluation of antigen analytes, such as proteins. In these methods a single chromogen is used to stain the sample. Sites of high analyte density will show a darker reaction and sites of low analyte density will show a weaker reaction. However, there is only a single color, light or dark, generated. Unfortunately, the analyte density in the sample is not easily evaluated by direct observation of laboratory personnel because only a single color is generated, and the human eye cannot distinguish between shades of color as easily as it can distinguish between different colors. Therefore, current methods of determining analyte density in a sample require the use of an image analysis system which records a digital image of the stained sample. The digital image is then converted to gray scale and the pixel density is measured from 0-255. Since gray-scale measurements typically use a range of 0-255, measurements of analyte density are limited to being described by this scale.
Therefore a need exists for novel methods for density measurement of target analytes in biological samples by differential staining. A further need exists for novel methods for density measurement of target analytes in biological samples which are easily evaluated by direct observation of laboratory personnel. There is also a need for novel methods for density measurement of target analytes in biological samples which do not require the use of an image analysis system. Furthermore, a need exists for novel methods for density measurement of target analytes in biological samples which do not limit descriptions of the analyte density to a scale of 0-255.